Introduction of labeled proteins into cells is essential for many situations in single molecule tracking. The existing methods involve perturbation of the cells, genetically encoded systems, or import mechanisms that leave the protein trapped in endosomes. A recent discovery published by the PI and his the two investigators on this grant, highlights how a peptide called Pep-1, and two Arg9 systems can be used to deliver proteins into the cytosol of COS-7 cells without capture into endosomes. They key is that the import was performed at 4[unreadable]C, where endosome formation is suppressed. This proposal is to develop this discovery into a practical, robust system for delivery of proteins into cells without endosomyl entrapment. Key steps in the project include: (i) establishing generality with respect to a range of proteins in a diverse set of cell lines;(ii) systematically preparing and testing analogs of the delivery systems to arrive at optimized vehicles that are tested in progressively more rigorous experiments for free functional protein;and, (iii) mechanistic studies to allow the structures of the delivery agents to be correlated with their functions. PUBLIC HEALTH RELEVANCE: This research is to develop a system that allows labeled proteins to be imported into cells without dramatically perturbing either the protein or the cell. This would be the first robust reagent of its kind, and would open the doors to single molecule tracking of proteins in living cells.